Hong CS , Saint-Jeannet JP .

R01 DE014212 NIDCR NIH HHS, R01 DE025468 NIDCR NIH HHS

             rhombomere morphogenesis
             hindbrain development

	Figure 2. Developmental expression of znf703. (a–d) By in situ hybridization at the neurula stage (NF stage 13/14) znf703 is detected in the dorso-lateral ectoderm posteriorly, including the neural plate and the neural crest forming region, with a gap of expression at the dorsal midline. (e and f) At stages 16 through 19, znf703 expression persists in the dorsal ectoderm where it appears to be confined to the prospective hindbrain and spinal cord. (g) Transverse section of a stage 16 embryo, dorsal to top, showing znf703 expression in the neural plate (np), neural crest (nc), ventral non-neural ectoderm (nne) and lateral plate mesoderm (lpm). (k–m) Later in development znf703 is diffusely expressed throughout the ectoderm, and enriched in the hatching gland (hg) and the branchial arches (ba). (a, c, e, h, and k) dorsal views, anterior to top. (b, d, j, l, and m) lateral views, dorsal to top, anterior to right. (f, and i) frontal views, dorsal to top. The embryonic stages are indicated in the lower left corner of each panel
	Figure 3. znf703 anterior expression domain corresponds to r3/r4. (a) At stage 15, znf703 is detected dorsally, with a sharp anterior boundary of expression. (b–d) Double in situ hybridization showing the spatial relationship of znf703 with snai2 in the neural crest (b), mafb in r5/r6 (c), and fgf3 in r3/r4 (d) Dorsal views, anterior to top. The yellow lines indicate the position of the most anterior boundary of znf703 expression. The red asterisks indication the position of the most anterior expression domains of snai2, mafb, and fgf3. Insets (lower right corner) in panels (b), (e), and (d) depict single in situ hybridization for snai2, mafb, and fgf3, respectively
	Figure 4. znf703 is a true target of Pax3 and Zic1. (a and b) Fold induction of snai2 (a) and znf703 (b) by expression of pax3GR and zic1GR in animal cap explants (microarray data; Bae et al., 2014). (c and d) Injection in one blastomere at the two-cell stage of MOs blocking Pax3 (pax3MO; 40 ng) Zic1 (zic1MO; 40 ng) function resulted in a strong reduction of snai2 expression (c),r while only the most anterior expression domain of znf703 was affected (d) at the neurula stage. The injected side is to the right as indicated by the presence of the lineage tracer (Red-Gal). The percentage of affected embryos is indicated in the upper right corner. Dorsal views, anterior to top.
	Figure 5. Regulation of znf703 expression by neural crest inducing signals. (a) mRNAs encoding noggin (1 ng) and wnt1 (100 pg) were injected, alone or in combination with pax3MO, into both blastomeres in the animal pole region at the 2-cell stage. At the blastula stage (NF Stage 9), animal cap explants were dissected, cultured for 8 hr at room temperature (equivalent Stage 13/14) and analyzed by qRT-PCR. (b) In combination, Noggin and Wnt1 induce snai2 and repress keratin expression. Co-injection of pax3MO blocks snai2 induction by Noggin and Wnt1 and promotes neural fate (sox2 expression). The induction of znf703 by Noggin and Wnt1 is unaffected by injection of pax3MO
	Figure 6. Znf703 is required for posterior hindbrain patterning. (a) The target sequence of Znf703 morpholino antisense oligonucleotide (Znf703MO; grey box) is indicated. The position of the start codon (ATG) is indicated. (b) In vitro coupled transcription/translation reactions with plasmid encoding Znf703. Increasing amounts of Znf703MO, 10 ng (+), 100 ng (++) and 1000 ng (+++), block translation directed by znf703 mRNA. (c–j) Unilateral injection of znf703MO (45 ng) at the two-cell stage expanded egr4 (c), hoxb1 (d) and fgf3 (e) expression domains in the hindbrain at the neurula stage (NF Stage 14/15). In contrast mafb (f) and the r5 expression domain of egr2 were reduced or lost (g). In morphant embryos the expression domain of sox2 was expanded (h), while the neural crest markers were mildly (snai2; g) or strongly (sox10; h) reduced. The horizontal lines (f) indicate the width of the neural plate on the control (black line) and injected (white line) sides. The injected side is to the right as indicated by the presence of the lineage tracer (Red-Gal). The percentage of affected embryos is indicated in the upper right corner. Dorsal views, anterior to top
	Figure 7. Znf703 overexpression affects hindbrain and neural crest development. (a–h) Unilateral injection of znf703 mRNA (1 ng) at the two-cell stage significantly reduced the expression domain of egr4 (a), hoxb1 (b), fgf3 (c) mafb (d), and egr2 (e), in the hindbrain at the neurula stage (NF Stage 14/15). In these embryos sox2 expression domain was expanded (f), and both neural crest markers, snai2 (g) and sox10 (h), were strongly reduced. The horizontal lines (f) indicate the width of the neural plate on the control (black line) and injected (white line) sides. The injected side is to the right as indicated by the presence of the lineage tracer (Red-Gal). The percentage of affected embryos is indicated in the upper right corner. Dorsal views, anterior to top
	znf703 (zinc finger protein 703) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 13, dorsal view, anterior up.
	znf703 (zinc finger protein 703) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 13, lateral view, anterior right, dorsal up.
	znf703 (zinc finger protein 703) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 14, lateral view, anterior right, dorsal up.
	znf703 (zinc finger protein 703) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 16, (e) dorsal view, anterior up and (f) anterior view,, dorsal up.
	znf703 (zinc finger protein 703) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 19 (i), anterior view, dorsal up and (j) lateral view, anterior right, dorsal up.
	znf703 (zinc finger protein 703) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 25, dorsal view, anterior up.
	znf703 (zinc finger protein 703) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 25, anterior/lateral view, dorsal up.
	znf703 (zinc finger protein 703) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 25, lateral view, anterior right, dorsal up.
	znf703 (zinc finger protein 703) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 16, transverse section, dorsal up, showing expression in the non-neural ectoderm (nne) and the underlying lateral plate mesoderm (lpm), as well as neural crest ( nc) and neural plate (np)

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